Adjusting Catalytic Activity of ß-Amyrin Synthase GgBAS by Utilizing the Plasticity Residues of an Active Site.

ß-Amyrin synthase (bAS) is a representative plant oxidosqualene cyclase (OSC), and previous studies have identified many functional residues and mutants that can alter its catalytic activity. However, the regulatory mechanism of the active site architecture for adjusting the catalytic activity remains unclear. In this study, we investigate the function of key residues and their regulatory effects on the catalytic activity of Glycyrrhiza glabra ß-amyrin synthase (GgbAS) through molecular dynamics simulations and site-directed mutagenesis experiments. We identified the plasticity residues located in two active site regions and explored the interactions between these residues and tetracyclic/pentacyclic intermediates. Based on computational and experimental results, we further categorize these plasticity residues into three types: effector, adjuster, and supporter residues, according to their functions in the catalytic process. This study provides valuable insights into the catalytic mechanism and active site plasticity of GgbAS, offering important references for the rational enzyme engineering of other OSC enzyme.

Genome-Wide Investigation of Oxidosqualene Cyclase Genes Deciphers the Genetic Basis of Triterpene Biosynthesis in Tea Plants.

Triterpenoids from Camellia species comprise a diverse class of bioactive compounds with great therapeutic potential. However, triterpene biosynthesis in tea plants (Camellia sinensis) remains elusive. Here, we identified eight putative 2,3-oxidosqualene cyclase (OSC) genes (CsOSC1-8) from the tea genome and characterized the functions of five through heterologous expression in yeast and tobacco and transient overexpression in tea plants. CsOSC1 was found to be a ß-amyrin synthase, whereas CsOSC4, 5, and 6 exhibited multifunctional α-amyrin synthase activity. Molecular docking and site-directed mutagenesis showed that the CsOSC6M259T/W260L double mutant yielded >40% lupeol, while the CsOSC1 W259L single mutant alone was sufficient for lupeol production. The V732F mutation in CsOSC5 altered product formation from friedelin to taraxasterol and ψ-taraxasterol. The L254 M mutation in the cycloartenol synthase CsOSC8 enhanced the catalytic activity. Our findings shed light on the molecular basis governing triterpene diversity in tea plants and offer potential avenues for OSC engineering.

Characterization of a Structurally Distinct ATP-Dependent Reactivating Factor of Adenosylcobalamin-Dependent Lysine 5,6-Aminomutase.

Several anaerobic bacterial species, including the Gram-negative oral bacterium Fusobacterium nucleatum, ferment lysine to produce butyrate, acetate, and ammonia. The second step of the metabolic pathway─isomerization of ß-l-lysine to erythro-3,5-diaminohexanoate─is catalyzed by the adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP)-dependent enzyme, lysine 5,6-aminomutase (5,6-LAM). Similar to other AdoCbl-dependent enzymes, 5,6-LAM undergoes mechanism-based inactivation due to loss of the AdoCbl 5'-deoxyadenosyl moiety and oxidation of the cob(II)alamin intermediate to hydroxocob(III)alamin. Herein, we identified kamB and kamC, two genes responsible for ATP-dependent reactivation of 5,6-LAM. KamB and KamC, which are encoded upstream of the genes corresponding to α and ß subunits of 5,6-LAM (kamD and kamE), co-purified following coexpression of the genes in Escherichia coli. KamBC exhibited a basal level of ATP-hydrolyzing activity that was increased 35% in a reaction mixture that facilitated 5,6-LAM turnover with ß-l-lysine or d,l-lysine. Ultraviolet-visible (UV-vis) spectroscopic studies performed under anaerobic conditions revealed that KamBC in the presence of ATP/Mg2+ increased the steady-state concentration of the cob(II)alamin intermediate in the presence of excess ß-l-lysine. Using a coupled UV-visible spectroscopic assay, we show that KamBC is able to reactivate 5,6-LAM through exchange of the damaged hydroxocob(III)alamin for AdoCbl. KamBC is also specific for 5,6-LAM as it had no effect on the rate of substrate-induced inactivation of the homologue, ornithine 4,5-aminomutase. Based on sequence homology, KamBC is structurally distinct from previously characterized B12 chaperones and reactivases, and correspondingly adds to the list of proteins that have evolved to maintain the cellular activity of B12 enzymes.

Recent developments, opportunities, and challenges in the study of mRNA pseudouridylation.

Pseudouridine is an abundant mRNA modification found in diverse organisms ranging from bacteria and viruses to multicellular plants and humans. New developments in pseudouridine profiling provide quantitative tools to map mRNA pseudouridylation sites. Sparse biochemical studies establish the potential for mRNA pseudouridylation to affect most stages of the mRNA life cycle from birth to death. This recent progress sets the stage for deeper investigations into the molecular and cellular functions of specific mRNA pseudouridines, including in disease.

Landscape of RNA pseudouridylation in archaeon Sulfolobus islandicus.

Pseudouridine, one of the most abundant RNA modifications, is synthesized by stand-alone or RNA-guided pseudouridine synthases. Here, we comprehensively mapped pseudouridines in rRNAs, tRNAs and small RNAs in the archaeon Sulfolobus islandicus and identified Cbf5-associated H/ACA RNAs. Through genetic deletion and in vitro modification assays, we determined the responsible enzymes for these modifications. The pseudouridylation machinery in S. islandicus consists of the stand-alone enzymes aPus7 and aPus10, and six H/ACA RNA-guided enzymes that account for all identified pseudouridines. These H/ACA RNAs guide the modification of all eleven sites in rRNAs, two sites in tRNAs, and two sites in CRISPR RNAs. One H/ACA RNA shows exceptional versatility by targeting eight different sites. aPus7 and aPus10 are responsible for modifying positions 13, 54 and 55 in tRNAs. We identified four atypical H/ACA RNAs that lack the lower stem and the ACA motif and confirmed their function both in vivo and in vitro. Intriguingly, atypical H/ACA RNAs can be modified by Cbf5 in a guide-independent manner. Our data provide the first global view of pseudouridylation in archaea and reveal unexpected structures, substrates, and activities of archaeal H/ACA RNPs.

The galactose metabolism genes UGE1 and UGM1 are novel virulence factors of the maize anthracnose fungus Colletotrichum graminicola.

Fungal cell walls represent the frontline contact with the host and play a prime role in pathogenesis. While the roles of the cell wall polymers like chitin and branched ß-glucan are well understood in vegetative and pathogenic development, that of the most prominent galactose-containing polymers galactosaminogalactan and fungal-type galactomannan is unknown in plant pathogenic fungi. Mining the genome of the maize pathogen Colletotrichum graminicola identified the single-copy key galactose metabolism genes UGE1 and UGM1, encoding a UDP-glucose-4-epimerase and UDP-galactopyranose mutase, respectively. UGE1 is thought to be required for biosynthesis of both polymers, whereas UGM1 is specifically required for fungal-type galactomannan formation. Promoter:eGFP fusion strains revealed that both genes are expressed in vegetative and in pathogenic hyphae at all stages of pathogenesis. Targeted deletion of UGE1 and UGM1, and fluorescence-labeling of galactosaminogalactan and fungal-type galactomannan confirmed that Δuge1 mutants were unable to synthesize either of these polymers, and Δugm1 mutants did not exhibit fungal-type galactomannan. Appressoria of Δuge1, but not of Δugm1 mutants, were defective in adhesion, highlighting a function of galactosaminogalactan in the establishment of these infection cells on hydrophobic surfaces. Both Δuge1 and Δugm1 mutants showed cell wall defects in older vegetative hyphae and severely reduced appressorial penetration competence. On intact leaves of Zea mays, both mutants showed strongly reduced disease symptom severity, indicating that UGE1 and UGM1 represent novel virulence factors of C. graminicola.

Bacteria at Work - Experimental and Theoretical Studies Reveal the Catalytic Mechanism of Ectoine Synthase.

Ectoine synthase (EctC) catalyses the ultimate step of ectoine biosynthesis, a kosmotropic compound produced as compatible solute by many bacteria and some archaea or eukaryotes. EctC is an Fe2+-dependent homodimeric cytoplasmic protein. Using Mössbauer spectroscopy, molecular dynamics simulations and QM/MM calculations, we determined the most likely coordination number and geometry of the Fe2+ ion and proposed a mechanism of the EctC-catalysed reaction. Most notably, we show that apart from the three amino acids binding to the iron ion (Glu57, Tyr84 and His92), one water molecule and one hydroxide ion are required as additional ligands for the reaction to occur. They fill the first coordination sphere of the Fe2+-cofactor and act as critical proton donors and acceptors during the cyclization reaction.

Chromosome-level genome assembly of Prunella vulgaris L. provides insights into pentacyclic triterpenoid biosynthesis.

Prunella vulgaris is one of the bestselling and widely used medicinal herbs. It is recorded as an ace medicine for cleansing and protecting the liver in Chinese Pharmacopoeia and has been used as the main constitutions of many herbal tea formulas in China for centuries. It is also a traditional folk medicine in Europe and other countries of Asia. Pentacyclic triterpenoids are a major class of bioactive compounds produced in P. vulgaris. However, their biosynthetic mechanism remains to be elucidated. Here, we report a chromosome-level reference genome of P. vulgaris using an approach combining Illumina, ONT, and Hi-C technologies. It is 671.95 Mb in size with a scaffold N50 of 49.10 Mb and a complete BUSCO of 98.45%. About 98.31% of the sequence was anchored into 14 pseudochromosomes. Comparative genome analysis revealed a recent WGD in P. vulgaris. Genome-wide analysis identified 35 932 protein-coding genes (PCGs), of which 59 encode enzymes involved in 2,3-oxidosqualene biosynthesis. In addition, 10 PvOSC, 358 PvCYP, and 177 PvUGT genes were identified, of which five PvOSCs, 25 PvCYPs, and 9 PvUGTs were predicted to be involved in the biosynthesis of pentacyclic triterpenoids. Biochemical activity assay of PvOSC2, PvOSC4, and PvOSC6 recombinant proteins showed that they were mixed amyrin synthase (MAS), lupeol synthase (LUS), and ß-amyrin synthase (BAS), respectively. The results provide a solid foundation for further elucidating the biosynthetic mechanism of pentacyclic triterpenoids in P. vulgaris.

Cloning and functional characterization of the oxidative squalene cyclase gene in the deep-sea holothurian Chiridota sp.

Saponins derived from holothurians have high potential medicinal value. However, the de novo synthesis of the derivatization of triterpenes is still unclear. Oxidative squalene cyclase (OSC) can catalyze 2,3-Oxidosqualene into diverse products that serve as important precursors for triterpene synthesis. However, the function of theOSCgene in Chiridotasp. hasnot been elucidated. In this study, an OSCgenederived from the deep-sea holothurianChiridota sp. was cloned and characterized functionally in a yeast system. The open reading frame of the OSC gene was 2086 bp, which encoded 695 amino acids. The Chiridota sp. OSC gene has a similarity of 66.89 % to the OSC of other holothurian species and 63.51 % to that of Acanthaster planci. The phylogenetic tree showed that the echinozoan OSCsclustered together, and then they formeda sister group to fungi and plant homologs. Chiridota sp. OSC catalyzed 2,3-Oxidosqualene into parkeol.Under high pressure, the relative enzymatic activity and stability of cyclase inChiridota sp. was higher than that in the shallow-sea holothurianStichopus horrens. The newly cloned OSC of Chiridota sp.provideskey information for the interpretation of the saponin synthesis pathway in deep-sea holothurians.

Insight into neofunctionalization of 2,3-oxidosqualene cyclases in B,C-ring-opened triterpene biosynthesis in quinoa.

Bioactive triterpenes feature complex fused-ring structures, primarily shaped by the first-committed enzyme, 2,3-oxidosqualene cyclases (OSCs) in plant triterpene biosynthesis. Triterpenes with B,C-ring-opened skeletons are extremely rare with unknown formation mechanisms, harbouring unchartered chemistry and biology. Here, through mining the genome of Chenopodium quinoa followed by functional characterization, we identified a stress-responsive and neofunctionalized OSC capable of generating B,C-ring-opened triterpenes, including camelliol A and B and the novel (-)-quinoxide A as wax components of the specialized epidermal bladder cells, namely the quinoxide synthase (CqQS). Protein structure analysis followed by site-directed mutagenesis identified key variable amino acid sites underlying functional interconversion between pentacyclic ß-amyrin synthase (CqbAS1) and B,C-ring-opened triterpene synthase CqQS. Mutation of one key residue (N612K) in even evolutionarily distant Arabidopsis ß-amyrin synthase could generate quinoxides, indicating a conserved mechanism for B,C-ring-opened triterpene formation in plants. Quantum computation combined with docking experiments further suggests that conformations of conserved W613 and F413 of CqQS might be key to selectively stabilizing intermediate carbocations towards B,C-ring-opened triterpene formation. Our findings shed light on quinoa triterpene skeletal diversity and mechanisms underlying B,C-ring-opened triterpene biosynthesis, opening avenues towards accessing their chemistry and biology and paving the way for quinoa trait engineering and quality improvement.

OXIDOSQUALENE CYCLASE 1 and 2 influence triterpene biosynthesis and defense in Nicotiana attenuata.

Triterpenes are a class of bioactive compounds with diverse biological functions, playing pivotal roles in plant defense against biotic stressors. Oxidosqualene cyclases (OSCs) serve as gatekeepers in the biosynthesis of triterpenes. In this study, we utilized a Nicotiana benthamiana heterologous expression system to characterize NaOSC1 from Nicotiana attenuata as a multifunctional enzyme capable of synthesizing lupeol, dammarenediol II, 3-alpha,20-lupanediol, and 7 other triterpene scaffolds. We also demonstrated that NaOSC2 is, in contrast, a selective enzyme, producing only the ß-amyrin scaffold. Through virus-induced gene silencing and in vitro toxicity assays, we elucidated the roles of NaOSC1 and NaOSC2 in the defense of N. attenuata against Manduca sexta larvae. Metabolomic and feature-based molecular network analyses of leaves with silenced NaOSC1 and NaOSC2 unveiled 3 potential triterpene glycoside metabolite clusters. Interestingly, features identified as triterpenes within these clusters displayed a significant negative correlation with larval mass. Our study highlights the pivotal roles of NaOSC1 and NaOSC2 from N. attenuata in the initial steps of triterpene biosynthesis, subsequently influencing defense against M. sexta through the modulation of downstream triterpene glycoside compounds.

The structural basis of mRNA recognition and binding by yeast pseudouridine synthase PUS1.

The chemical modification of RNA bases represents a ubiquitous activity that spans all domains of life. Pseudouridylation is the most common RNA modification and is observed within tRNA, rRNA, ncRNA and mRNAs. Pseudouridine synthase or 'PUS' enzymes include those that rely on guide RNA molecules and others that function as 'stand-alone' enzymes. Among the latter, several have been shown to modify mRNA transcripts. Although recent studies have defined the structural requirements for RNA to act as a PUS target, the mechanisms by which PUS1 recognizes these target sequences in mRNA are not well understood. Here we describe the crystal structure of yeast PUS1 bound to an RNA target that we identified as being a hot spot for PUS1-interaction within a model mRNA at 2.4 Å resolution. The enzyme recognizes and binds both strands in a helical RNA duplex, and thus guides the RNA containing the target uridine to the active site for subsequent modification of the transcript. The study also allows us to show the divergence of related PUS1 enzymes and their corresponding RNA target specificities, and to speculate on the basis by which PUS1 binds and modifies mRNA or tRNA substrates.

Structure-Guided Discovery of N5-CAIR Mutase Inhibitors.

Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of antimicrobial therapies. While humans biosynthesize purine nucleotides in a 10-step pathway, most microbes utilize an additional 11th enzymatic activity. The human enzyme, aminoimidazole ribonucleotide (AIR) carboxylase generates the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) directly. Most microbes, however, require two separate enzymes, a synthetase (PurK) and a mutase (PurE), and proceed through the intermediate, N5-CAIR. Toward the development of therapeutics that target these differences, we have solved crystal structures of the N5-CAIR mutase of the human pathogens Legionella pneumophila (LpPurE) and Burkholderia cenocepacia (BcPurE) and used a structure-guided approach to identify inhibitors. Analysis of the structures reveals a highly conserved fold and active site architecture. Using this data, and three additional structures of PurE enzymes, we screened a library of FDA-approved compounds in silico and identified a set of 25 candidates for further analysis. Among these, we identified several new PurE inhibitors with micromolar IC50 values. Several of these compounds, including the α1-blocker Alfuzosin, inhibit the microbial PurE enzymes much more effectively than the human homologue. These structures and the newly described PurE inhibitors are valuable tools to aid in further studies of this enzyme and provide a foundation for the development of compounds that target differences between human and microbial purine metabolism.

The 2-methylpropene degradation pathway in Mycobacteriaceae family strains.

Mycolicibacterium gadium IBE100 and Mycobacterium paragordonae IBE200 are aerobic, chemoorganoheterotrophic bacteria isolated from activated sludge from a wastewater treatment plant. They use 2-methylpropene (isobutene, 2-MP) as the sole source of carbon and energy. Here, we postulate a degradation pathway of 2-methylpropene derived from whole genome sequencing, differential expression analysis and peptide-mass fingerprinting. Key genes identified are coding for a 4-component soluble diiron monooxygenase with epoxidase activity, an epoxide hydrolase, and a 2-hydroxyisobutyryl-CoA mutase. In both strains, involved genes are arranged in clusters of 61.0 and 58.5 kbp, respectively, which also contain the genes coding for parts of the aerobic pathway of adenosylcobalamin synthesis. This vitamin is essential for the carbon rearrangement reaction catalysed by the mutase. These findings provide data for the identification of potential 2-methylpropene degraders.

Aging-induced pseudouridine synthase 10 impairs hematopoietic stem cells.

Aged hematopoietic stem cells (HSC) exhibit compromised reconstitution capacity and differentiation-bias towards myeloid lineage, however, the molecular mechanism behind it remains not fully understood. In this study, we observed that the expression of pseudouridine (Ψ) synthase 10 is increased in aged hematopoietic stem and progenitor cells (HSPC) and enforced protein of Ψ synthase 10 (PUS10) recapitulates the phenotype of aged HSC, which is not achieved by its Ψ synthase activity. Consistently, we observed no difference of transcribed RNA pseudouridylation profile between young and aged HSPC. No significant alteration of hematopoietic homeostasis and HSC function is observed in young Pus10-/- mice, while aged Pus10-/- mice exhibit mild alteration of hematopoietic homeostasis and HSC function. Moreover, we observed that PUS10 is ubiquitinated by E3 ubiquitin ligase CRL4DCAF1 complex and the increase of PUS10 in aged HSPC is due to aging-declined CRL4DCAF1- mediated ubiquitination degradation signaling. Taken together, this study for the first time evaluated the role of PUS10 in HSC aging and function, and provided a novel insight into HSC rejuvenation and its clinical application.

Harnessing the Structure and Dynamics of the Squalene-Hopene Cyclase for (-)-Ambroxide Production.

Terpene cyclases offer enormous synthetic potential, given their unique ability to forge complex hydrocarbon scaffolds from achiral precursors within a single cationic rearrangement cascade. Harnessing their synthetic power, however, has proved to be challenging owing to their generally low catalytic performance. In this study, we unveiled the catalytic potential of the squalene-hopene cyclase (SHC) by harnessing its structure and dynamics. First, we synergistically tailored the active site and entrance tunnel of the enzyme to generate a 397-fold improved (-)-ambroxide synthase. Our computational investigations explain how the introduced mutations work in concert to improve substrate acquisition, flow, and chaperoning. Kinetics, however, showed terpene-induced inactivation of the membrane-bound SHC to be the major turnover limitation in vivo. Merging this insight with the improved and stereoselective catalysis of the enzyme, we applied a feeding strategy to exceed 105 total turnovers. We believe that our results may bridge the gap for broader application of SHCs in synthetic chemistry.

The Identification of SQS/SQE/OSC Gene Families in Regulating the Biosynthesis of Triterpenes in Potentilla anserina.

The tuberous roots of Potentilla anserina (Pan) are an edible and medicinal resource in Qinghai-Tibetan Plateau, China. The triterpenoids from tuberous roots have shown promising anti-cancer, hepatoprotective, and anti-inflammatory properties. In this study, we carried out phylogenetic analysis of squalene synthases (SQSs), squalene epoxidases (SQEs), and oxidosqualene cyclases (OSCs) in the pathway of triterpenes. In total, 6, 26, and 20 genes of SQSs, SQEs, and OSCs were retrieved from the genome of Pan, respectively. Moreover, 6 SQSs and 25 SQEs genes expressed in two sub-genomes (A and B) of Pan. SQSs were not expanded after whole-genome duplication (WGD), and the duplicated genes were detected in SQEs. Twenty OSCs were divided into two clades of cycloartenol synthases (CASs) and ß-amyrin synthases (ß-ASs) by a phylogenetic tree, characterized with gene duplication and evolutionary divergence. We speculated that ß-ASs and CASs may participate in triterpenes synthesis. The data presented act as valuable references for future studies on the triterpene synthetic pathway of Pan.

De Novo Transcriptome Sequencing of Codonopsis lanceolata for Identification of Triterpene Synthase and Triterpene Acetyltransferase.

Codonopsis lanceolata (Campanulaceae) is a perennial plant commonly known as the bonnet bellflower. This species is widely used in traditional medicine and is considered to have multiple medicinal properties. In this study, we found that shoots and roots of C. lanceolata contained various types of free triterpenes (taraxerol, ß-amyrin, α-amyrin, and friedelin) and triterpene acetates (taraxerol acetate, ß-amyrin acetate, and α-amyrin acetate). The content of triterpenes and triterpene acetates by GC analysis was higher in the shoot than in the roots. To investigate the transcriptional activity of genes involved in triterpenes and triterpene acetate biosynthesis, we performed de novo transcriptome analysis of shoots and roots of C. lanceolata by sequencing using the Illumina platform. A total of 39,523 representative transcripts were obtained. After functional annotation of the transcripts, the differential expression of genes involved in triterpene biosynthetic pathways was investigated. Generally, the transcriptional activity of unigenes in the upstream region (MVA and MEP pathway) of triterpene biosynthetic pathways was higher in shoots than in roots. Various triterpene synthases (2,3-oxidosqualene cyclase, OSC) participate to produce triterpene skeletons by the cyclization of 2,3-oxidosqualene. A total of fifteen contigs were obtained in annotated OSCs in the representative transcripts. Functional characterization of four OSC sequences by heterologous expression in yeast revealed that ClOSC1 was determined as taraxerol synthase, and ClOSC2 was a mixed-amyrin synthase producing α-amyrin and ß-amyrin. Five putative contigs of triterpene acetyltransferases showed high homology to the lettuce triterpene acetyltransferases. Conclusively, this study provides the basis of molecular information, particularly for the biosynthesis of triterpenes and triterpene acetates in C. lanceolata.

Importance of aspartic acid side chain carboxylate-arginine interaction in substrate selection of arginine 2,3-aminomutase BlsG.

The fungicide nucleoside blasticidin S features a ß-arginine, a moiety seldom revealed in the structure of natural products. BlsG, a radical SAM arginine-2,3-aminomutase from the blasticidin S biosynthetic pathway, displayed promiscuous activity to three basic amino acids. Here in this study, we demonstrated that BlsG showed high preference toward its natural substrate arginine. The combined structural modeling, steady-state kinetics, and mutational analyses lead to the detailed understanding of the substrate recognition of BlsG. A single mutation of T340D changed the substrate preference of BlsG leading to a little more preference to lysine than arginine. On the basis of our understanding of the substrate selection of BlsG and bioinformatic analysis, we propose that the D…D motif locationally corresponding to D293 and D330 of KAM is characteristic of lysine 2,3-aminomutase while the corresponding D…T motif is characteristic of arginine 2,3-aminomutase. The study may provide a simple way to discern the arginine 2,3-aminomutase and thus lead to the discovery of new natural compounds with ß-arginine moiety.

UDP-Galactopyranose Mutase Mediates Cell Wall Integrity, Polarity Growth, and Virulence in Fusarium graminearum.

CHY1 is a zinc finger protein unique to microorganisms that was found to regulate polarized tip growth in Fusarium graminearum, an important pathogen of wheat and barley. To further characterize its functions, in this study we identified CHY1-interacting proteins by affinity purification and selected UDP-galactofuranose (Galf) mutase (UGMA) for detailed characterization, because UGMA and UDP-Galf are unique to fungi and bacteria and absent in plants and animals. The interaction between CHY1 and UGMA was confirmed by yeast two-hybrid assays. Deletion of UGMA in F. graminearum resulted in significant defects in vegetative growth, reproduction, cell wall integrity, and pathogenicity. Infection with the ΔugmA mutant was restricted to the inoculated floret, and no vomitoxin was detected in kernels inoculated with the ΔugmA strain. Compared to the wild type, the ΔugmA mutant produced wide, highly branched hyphae with thick walls, as visualized by transmission electron microscopy. UGMA tagged with green fluorescent protein (GFP) mainly localized to the cytoplasm, consistent with the synthesis of Galf in the cytoplasm. The Δchy1 mutant was more sensitive, while the ΔugmA mutant was more tolerant, to cell wall-degrading enzymes. The growth of the ΔugmA mutant nearly ceased upon caspofungin treatment. More interestingly, nocodazole treatment of the ΔugmA strain attenuated its highly branched morphology, while caspofungin inhibited the degree of the twisted Δchy1 mycelia, indicating that CHY1 and UGMA probably have opposite effects on cell wall architecture. In conclusion, UGMA is an important pathogenic factor that is specific to fungi and bacteria and required for cell wall architecture, radial growth, and caspofungin tolerance, and it appears to be a promising target for antifungal agent development. IMPORTANCE The long-term use of chemical pesticides has had increasingly negative impacts on the ecological environment and human health. Low-toxicity, high-efficiency and environmentally friendly alternative pesticides are of great significance for maintaining the sustainable development of agriculture and human and environmental health. Using fungus- or microbe-specific genes as candidate targets provides a good foundation for the development of low-toxicity, environmentally friendly pesticides. In this study, we characterized a fungus- and bacterium-specific UDP-galactopyranose mutase gene, ugmA, that contributes to the synthesis of the cell wall component Galf and is required for vegetative growth, cell wall integrity, deoxynivalenol (DON) production, and pathogenicity in F. graminearum. The ugmA deletion mutant exhibited increased sensitivity to caspofungin. These results demonstrate the functional importance of UGMA in F. graminearum, and its absence from mammals and higher plants constitutes a considerable advantage as a low-toxicity target for the development of new anti-Fusarium agents.